Using an HPLC-Chip System with Trap XCT Ultra MS for the Identification and Relative

Application Note More than 90 % of the protein drugs in existence are glycoproteins. Research has shown that the efficacy of a glycoprotein drug depends critically on the type and extent of glycosylation of the protein. Consequently, it is critical to characterize glycoproteins as an essential part of drug quality control. This application note explores the ability of Agilent's HPLC-Chip/Trap XCT Ultra MS system to detect and identify oligosaccharides and N-linked glycans cleaved from bovine fetuin and human IgG. The HPLC-Chip used for these experiments contained a porous graphitized carbon column designed specifically for oligosaccharide separation applications. Analysis of a 10 fmol injection of a mixture of maltooligosaccharide alditols indicated that glycans present at the sub-picomole level are readily visible in the total ion chromatogram or base peak chromatogram (BPC). Released N-linked glycans from 5 pmol of bovine fetuin as starting protein were clearly identifiable in the BPC and produced clean MS/MS spectra. Analysis of N-linked glycans from IgG, using an on-column amount equivalent to glycans from 1 pmol starting glycoprotein, showed that the instrument has the requisite separation power and sensitivity to detect and identify even minor IgG variants. These results indicate that the combination of an Agilent HPLC-Chip equipped with a porous graphitized carbon nanocolumn and the Agilent Trap XCT Ultra LC/MSD system has sufficient sensitivity to analyze N-linked glycans released from picomole amounts of a starting glycoprotein, even permitting detection of minor glycoforms whose presence or absence may be significant in the stability or effectiveness of a glycoprotein drug